The nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is cytolytic when expressed in transformed cells. Before causing extensive cell lysis, NS1 induces a multistep cell cycle arrest in G 1 , S, and G 2 , well reproducing the arrest in S and G 2 observed upon MVMp infection. In this work we investigated the molecular mechanisms of growth inhibition mediated by NS1 and MVMp. We show that NS1-mediated cell cycle arrest correlates with the accumulation of the cyclin-dependent kinase (Cdk) inhibitor p21 cip1 associated with both the cyclin A/Cdk and cyclin E/Cdk2 complexes but in the absence of accumulation of p53, a potent transcriptional activator of p21 cip1 . By comparison, MVMp infection induced the accumulation of both p53 and p21 cip1 . We demonstrate that p53 plays an essential role in the MVMp-induced cell cycle arrest in both S and G 2 by using p53 wild-type (؉/؉) and null (؊/؊) cells. Furthermore, only the G 2 arrest was abrogated in p21 cip1 null (؊/؊) cells. Together these results show that the MVMp-induced cell cycle arrest in S is p53 dependent but p21 cip1 independent, whereas the arrest in G 2 depends on both p53 and its downstream effector p21 cip1 . They also suggest that induction of p21 cip1 by the viral protein NS1 arrests cells in G 2 through inhibition of cyclin A-dependent kinase activity.

The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct injection into the macronucleus of Paramecium tetraurelia of DNA molecules coding for the A-antigen leads to expression of the gene and autonomous replication. When injected into Paramecium primaurelia DNA from probably any origin, procaryote or eucaryote, can replicate as linear telomerized molecules and the number of copies maintained can be very high (up to 20000 copies). We present here evidence that if the injected linear DNA molecules harbour preexisting telomeres at both extremities they are protected from degradation, the number of DNA molecules maintained being 15- to 30-fold higher than if the molecules are injected without telomeres. Some of the injected molecules replicate as multimers, but, only when the fused ends are devoid of preexisting telomeric repeats.

DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules. In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process. The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences. However, insertion sites were not totally at random. Roughly 30% of the molecules were integrated next to or in telomeric repeats. These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position. We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5x10-4 and 3x10-5.

Background: Cell division or cytokinesis, which results from a series of events starting in metaphase, is the mechanism by which the mother cell cytoplasm is divided between the two daughter cells. Hence it is the final step of the cell division cycle. The aim of the present study was to demonstrate that mammalian cells undergoing cytokinesis can be sorted selectively by flow cytometry. Materials and Methods: Cultures of HeLa cells were arrested in prometaphase by nocodazole, collected by mitotic shake-off and released for 90 min into fresh medium to enrich for cells undergoing cytokinesis. After ethanol fixation and DNA staining, cells were sorted based on DNA content and DNA fluorescence signal height.